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. 2010 Nov;21(11):1577-90.
doi: 10.1089/hum.2009.138. Epub 2010 Oct 19.

Unexpectedly high copy number of random integration but low frequency of persistent expression of the Sleeping Beauty transposase after trans delivery in primary human T cells

Xin Huang  1 , Kari Haley, Marianna Wong, Hongfeng Guo, Changming Lu, Andrew Wilber, Xianzheng Zhou
Affiliations

Unexpectedly high copy number of random integration but low frequency of persistent expression of the Sleeping Beauty transposase after trans delivery in primary human T cells

Xin Huang et al. Hum Gene Ther. 2010 Nov.

Abstract

We have shown that the Sleeping Beauty (SB) transposon system can mediate stable expression of both reporter and therapeutic genes in human primary T cells and that trans delivery (i.e., transposon and transposase are on separate plasmids) is at least 3-fold more efficient than cis delivery. One concern about trans delivery is the potential for integration of the transposase-encoding sequence into the cell genome with the possibility of continued expression, transposon remobilization, and insertional mutagenesis. To address this concern, human peripheral blood lymphocytes were nucleofected with transposase plasmid and a DsRed transposon. Eighty-eight stable DsRed(+) T cell clones were generated and found to be negative for the transposase-encoding sequence by PCR analysis of genomic DNA. Genomic PCR was positive for transposase in 5 of 15 bulk T cell populations that were similarly transfected and selected for transgene expression where copy numbers were unexpectedly high (0.007-0.047 per cell) by quantitative PCR. Transposase-positive bulk T cells lacked transposase plasmid demonstrated by Hirt (episomal) extracted DNA and showed no detectable transposase by Southern hybridization, Western blot, and quantitative RT-PCR analyses. Cytogenetic and array comparative genomic hybridization analyses of the only identified transposase-positive clone (O56; 0.867 copies per cell) showed no chromosomal abnormality or tumor formation in nude mice although transposon remobilization was detected. Our data suggest that SB delivery via plasmid in T cells should be carried out with caution because of unexpectedly high copy numbers of randomly integrated SB transposase.

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Figures

FIG. 1.
FIG. 1.
Confirmation of SB10 integration and expression in the O56 clone. (A) Genomic DNA analysis of SB10 expression in O56 and O69 T cell clones. SB10 plasmid DNA was used as a positive control. (B) Western blotting confirmation of SB10 expression in the O56 clone.
FIG. 2.
FIG. 2.
Determination of SB10 integration in multiple T cell clones. (A) Generation of T cell clones by FACS sorting of DsRed+ cells derived from SB-transfected PBLs. (B) Flow cytometric analysis of DsRed expression in T cell clones. Nineteen representative clones of 88 are shown. Nomenclature of each T cell clone was assigned as follows: The first number indicates the amount of transposase used for transfection; the second number indicates the plate number; the third number indicates the well position. (C) Genomic PCR analysis of SB10 expression in T cell clones. Seventeen of 88 clones are shown.
FIG. 3.
FIG. 3.
The SB transposon vectors used in this study. SB transposons contain inverted repeat/direct repeat sequences (IR/DR, indicated by arrowheads) flanking the gene of interest. CLP, CpG-less promoter; EF1α, a human elongation factor-1α promoter; PGK, human phosphoglycerate kinase promoter; Ubc, human ubiquitin C promoter; mC, minimal CMV promoter; Zeo, Zeocin resistance gene; GFP, green fluorescent protein; Bsd, blasticidin resistance gene; fLuc, firefly luciferase; NGCD, truncated human nerve growth factor receptor and cytosine deaminase fusion gene; 19BB, CD19 chimeric antigen receptor (CAR) with CD3ζ and 4-1BB signaling domain; gLuc, Gaussia luciferase; A, polyadenylation signal.
FIG. 4.
FIG. 4.
Molecular analysis of SB10 integration and expression in SB-transfected bulk T cells. (A) Genomic PCR showing that 5 of 15 bulk T cell populations transfected with SB10 or SB11 DNA plasmid were positive for transposase-encoding sequences. (B) Southern hybridization showing no detectable genomic integration of SB10 or SB11. (C) Standard curve of SB10 plasmid copy numbers by qPCR, where each point represents the mean ± SEM (n = 6 wells) of three independent assays. The standard curve for SB10 copy numbers was generated by dilution of the SB10 plasmid, ranging from 0, 0.004, 0.012, 0.037, 0.11, 0.33, 1, 3, to 9 copies. SB10 plasmid copy numbers less than 0.012 were considered not detectable and the copy number of 0.037 was not present on the linear line. Thus, the line equation established for the standard curve was based on the copy numbers ranging from 0.11, 0.33, 1, 3, to 9. (D) Copy numbers of the SB transposase in SB10+ or SB11+ bulk T cell lines and SB10+ clone O56. Data are presented as means ± SEM (n = 6 wells) of three independent assays. (E) RT-PCR showing no mRNA transcripts in genomic PCR-positive bulk T cells. (F) Quantitative RT-PCR showing undetectable mRNA transcript in SB10+ or SB11+ bulk T cells and an approximately 9000-fold higher expression level in O56 compared with O69. One of two representative results is shown. (G) Western blotting showing no SB10 or SB11 protein in genomic PCR-positive bulk T cells.
FIG. 5.
FIG. 5.
Characterization of transposon integrations in clones O56 and O69 cultured over time. (A) Linker-mediated PCR showing single bands in clone O56 and clone O69 cultured in the years 2004 and 2006. (B) Sequencing of PCR products showing mixed integrants in clone O56 but not in clone O69. Green highlights indicate the transposon sequence. Color images available online at www.liebertonline.com/hum.
FIG. 6.
FIG. 6.
Cytogenetic and array CGH analyses of O56 clone. (A) G-band analysis of SB10+ O56 and SB10– O69 clones showing a normal 46,XY male karyotype. (B) Array CGH showing losses at 7q34 and 14q11.2 in the control clone NO71 (compared with pooled male control specimens) and gains in the same regions in clones O56 and O69 (compared with NO71). (C) Array CGH showing copy number gains against control clone NO71 at 7q34 and 14q11.2 regions in clone O56. (D) Array CGH showing copy number gains against NO71 at 7q34 and 14q11.2 regions in clone O69. (E) The control NO71, compared with the pooled control DNA, showed two regions of loss encompassing approximately 346 and 155 kb within 7q34, respectively. The start point for the loss of the 346-kb region was estimated at bp 141663456 and the stop point at bp 142009059. The ratio value of −1 was consistent with a deletion of this region on one chromosome 7 allele. This region contains TCRBV genes. The start point for the loss of the 155-kb region was estimated at 142021348 and the stop point at 142176133. The ratio for this region fell between −2 and −4, consistent with a homozygous loss. Mapped to this region are other TCRB-related genes. Two regions of loss within 14q11.2, encompassing 128 and 633 kb, respectively, were noted. The start point for the 128-kb region was estimated at 21285126 and the stop point at 2142207. Mapped to this region are TCRAV-related genes. The start point for the 633-kb region was estimated at 21420105 and the stop point at 22052858. Mapped to this region are also TCRAV-related genes. Clone O56 shows a ratio of 1.0 for the proximal part of 7q34 and a ratio of 0 for the remaining portion of this 7q34 region. As the control clone NO71 had deletion in these regions, this would indicate that O56 had no loss for the proximal region, and had the same loss as the control NO71 for the distal portion of this region. Clone O69 had a small gain for a portion of the 7q34 region, likely indicating a mixture of cells that had the same loss as the control NO71 and those that had no loss. Clone O69 had a ratio of approximately 2.0 for the distal portion of 7q34, indicating no loss for this region. For the 14q11.2 region, the ratios for O56 and O69 were generated using the control T cell clone NO71, which showed a ratio of −3 for this region. Thus, the +0.3 ratio for O56 indicates that there is no loss for the proximal portion of 14q11.2 region in these cells; the remainder of this region shows a profile consistent with the presence of some cells with loss, and others not. The findings are similar for O69, which shows no loss for the very proximal region, and some cells with loss for the more distal portion of the region. Of note, for NO71 compared with pooled controls, additional gains were seen in the near centromeric regions of chromosomes 7, 8, and 15, and in the pseudoautosomal regions of Xp and Yp. These gains are among the benign copy number variants that are well documented in published databases to occur in healthy controls, and are interpreted as germline variants of no relevance to the present experiments. Color images available online at www.liebertonline.com/hum.
FIG. 7.
FIG. 7.
The O56 clone did not induce tumor formation in nude mice. (A) Tumor growth in nude mice; mice injected with the SB10+ O56 clone showed no tumor growth on day 73. In Raji cell-injected mice, one mouse (#1) showed initial tumor growth and then the tumor regressed spontaneously. (B) Tumor volume versus days after injection. Color images available online at www.liebertonline.com/hum.
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